Estrogens are steroid hormones that are essential for normal sexual development and functioning of female reproductive organs. Estrogens are also important for growth, differentiation, and functioning of the testis, epididymis and prostate in males. Estrogens also have important non-reproductive effects on bones and the heart. Estrogens comprise a group of natural and synthetic substances. Natural estrogens include estradiol (i.e., 17-β-estradiol or E2), estrone and estriol. Estrogens are sometimes given therapeutically in the form of a conjugate, such as for example, ethinyl estradiol, conjugated estrogens or diethylstilbestrol.
Tissues in the body that are responsive to estrogens are called “estrogen-sensitive” or “estrogen-responsive” tissues and include cells of the urogenital tract, cardiovascular system and skeletal system. The cells that comprise estrogen-sensitive tissues contain estrogen receptors (ER). ER can be of the α type or β type. Estrogens enter cells and bind to ER in the cytoplasm of such cells and an estrogen-ER complex is formed. Herein, a molecule such as estrogen that binds to a receptor is generally called a “ligand.” Herein, a receptor such as ER that has formed a complex with a ligand is called a “liganded” receptor.
Once the estrogen ligand binds to ER, the estrogen-ER complex migrates to the nucleus of the cell and binds to specific sequences of DNA within the cellular genome called “estrogen response elements.” Such estrogen response elements are located in the promoters of specific genes in the cell nucleus.
Binding of the estrogen-ER complex to estrogen-responsive elements causes activation or suppression of the transcription of the specific genes (Beato, et al., 1995, Cell, 83:851-7.; Katzenellenbogen, et al., 1995, J Steroid Biochem Mol Biol, 53:387-93.; Tsai and O'Malley, 1994, Annu Rev Biochem, 63:451-86.). The activation or suppression of specific gene transcription is one type of molecular and/or cellular response that can result from formation of a ligand-receptor complex. When such a response occurs, the receptor is said to have been “activated.”
Estrogen-ER complexes, therefore, act as transcription factors to regulate the expression of these genes. When a ligand binds to a receptor and a molecular and/or cellular response (e.g., transcriptional regulation of genes) occurs, such ligands are referred to as “agonists” and the response produced is called “agonism.” Herein, therefore, the term agonist refers to ligands, such as estrogen, that produce the molecular and/or cellular responses.
In addition to the role estrogens and ER play in normal development and functioning of cellular tissues, estrogens and ER play significant roles in certain human disease states, breast cancer being one specific example. Cells in female breast tissue normally contain ER. Interaction of estrogens with ER in breast cells normally causes the breasts to grow at puberty and again during pregnancy. Since breast cells normally contain ER, it is not surprising that cells comprising tumors of the breast also contain ER. Ninety-five percent of all breast tumors, at least initially, have ER and are dependent on estrogens for growth. In such breast tumor cells, estrogens acting via the ER, dramatically escalate proliferative and metastatic activity (Osborne, et al., 1980, Cancer, 46:2884-8.).
Treatment of such ER-positive breast tumors comprises administration to the individual with the tumor, compounds such as tamoxifen (TOT). TOT can also administered to individuals who may be at high risk for developing breast tumors in the future, for the purpose of prevention of such tumors. Chemically, tamoxifen is one of a number of compounds referred to as triphenyethylene derivatives. Tamoxifen is a mainstay of breast cancer treatment and inhibits the proliferation promoting effect of estrogens (Katzenellenbogen, et al., 1995, J Steroid Biochem Mol Biol, 53:387-93.; Osborne, et al., 1980, Cancer, 46:2884-8.; Jordan and Murphy, 1990, Endocr Rev, 11:578-610.). Like estrogens, TOT binds to ER and, therefore, is also an ER ligand. Unlike estrogen binding to ER, however, TOT binding to ER does not result in production of significant molecular and/or cellular responses. The changes in gene expression resulting from TOT binding to ER are significantly less in magnitude than those resulting from estrogen binding to ER. Such decreased responses are referred to as “partial agonism.” Ligands such as TOT, that result in partial agonism, are referred to as “partial agonists.”
Of significance is that binding of ER by TOT prevents estrogens from producing their effect on ER (i.e., the partial agonist precludes effects of the agonist). Since estrogens are prevented from producing a molecular and/or cellular response through the ER, the response produced in the presence of both estrogens and TOT will be partial agonism, rather than agonism. Such partial agonism is the basis by which TOT impairs breast tumor growth (i.e., by blocking the agonist effects of estrogens).
In addition to partial agonists like TOT, other substances exist that bind ER but then produce no molecular and/or cellular response. Such substances are referred to as “antagonists.” One such antagonist is ICI182,780 (ICI). ICI binding to ER prevents estrogens from binding to ER. Therefore, like TOT, ICI also impairs proliferation of ER-positive breast tumor cells caused by estrogen.
Substances that bind to the ER and prevent the molecular and/or cellular responses caused by estrogens are given the general name “selective estrogen receptor modulators” or SERMs (Osborne, et al., 2000, J Clin Oncol, 18:3172-86.). SERMs can also be called “antiestrogens.” SERMs encompass ER ligands that produce different responses. For example, one particular SERM may be an antagonist. Another particular SERM may be a partial agonist. Still another particular SERM may bind to ER and produce a molecular and/or cellular response that is only slightly less in magnitude than the response produced by estrogens. Such a SERM would result in a molecular and/or cellular response of a greater magnitude than the response produced by a partial agonist, but would not be referred to as an agonist because the molecular and/or cellular response is less than that produced by an agonist, like estrogen.
Chemically, SERMs can be classified into three groups (Osborne, et al., 2000, J Clin Oncol, 18:3172-86.). The first group comprises triphenylethylene derivatives, of which TOT is one. Other substances that are triphenylethylene derivatives are toremifene, droloxifene (3-hydroxytamoxifen), idoxifene, TAT-59 (a phosphorylated derivative of 4-hydroxytamoxifen) and GW5638 (a carboxylic acid derivative of tamoxifen). The second group of SERMs comprises other nonsteroidal compounds. This group comprises EM-800, EM-652 (benzopyran), raloxifene, LY353381 (SERM 3) and LY357489. The third group of SERMs comprises steroidal compounds that have a better ability to inhibit the response produced by estrogens. ICI182,780 (ICI) is a member of this third group. The listings of substances that comprise each group is not complete and others may exist.
With regard to TOT, while it is effective in preventing proliferation of ER-positive breast tumor cells (i.e., cells that contain ER) in the early stages of breast cancer treatment, such ER-positive tumor cells invariably develop resistance to TOT. That is, after a time (e.g., 5 years), TOT is no longer effective in preventing estrogen stimulation of tumor proliferation and, in fact, causes stimulation of proliferation of ER-positive tumor cells. This TOT-resistant phenotype of breast tumor cells cannot be attributed solely to a decrease or absence of ER expression in the cells since more that 70% of TOT-resistant tumors continue to express ER (Crawford, et al., 1987, Br J Cancer, 56:137-40.; Li, et al., 1994, J Surg Oncol, 57:71-7.).
There is a need to understand the mechanism by which cells respond to and become resistant to SERMs. There is particular need to understand how ER-positive tumor cells become resistant to the proliferation inhibiting effects of TOT. Understanding the mechanism of TOT resistance, for example, would provide methods for identifying, and perhaps for predicting which cells will become resistant to TOT. Understanding the mechanism of TOT resistance can also provide substances that inhibit development of TOT resistance. Such methods and substances can be used to enhance the beneficial effects and reduce the side effects of TOT therapy.